ENZYME CATALYSIS OF ELECTRON AND GROUP TRANSFER

电子和基团转移的酶催化

• 批准号: 2138202
• 负责人: PERRY A. FREY
• 金额: $ 20.26万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2138202
• 关键词: Clostridium; Escherichia coli; S adenosylmethionine; active sites; acyltransferase; chemical kinetics; cobalt; cofactor; deuterium; electron spin resonance spectroscopy; electron transport; enzyme complex; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate complex; fluorescence spectrometry; immobilized enzymes; iron sulfur protein; isomerase; nuclear magnetic resonance spectroscopy; pyridoxal phosphate; pyruvate dehydrogenase; thiamine pyrophosphate; ultraviolet spectrometry; vitamin B12 coenzyme

The long term objectives of the research supported by this grant are to determine the chemical mechanisms by which the coenzyme forms of vitamin B1 (thiamin pyrophosphate, TPP) and vitamin B6 (pyridoxal-5'-phosphate, PLP) act in reactions that cannot be explained by the conventional mechanisms known for these coenzyme. The role of 2-acetyl-TPP in the metabolism of pyruvate catalyzed by the pyruvate dehydrogenase (PDH) complex will be determined using chemical methods. Several inherited diseases of pyruvate metabolism are caused by defects in the PDH complex. The standard mechanistic paradigm for the coenzymatic action of PLP involves the stabilization of carbanionic intermediates by the coenzyme. However, in a major focus of research supported by this grant, the action of PLP in the lysine 2,3-aminomutase-catalyzed conversion of lysine of beta-lysine PLP is proposed to act by stabilizing a radical intermediate, rather than a carbanion,. This is a new chemical mechanism for PLP with other aminomutases, such as beta-lysine mutase and arginine 2,3,-aminomutase. The aminomutases catalyze 1,2-amino group migrations that are important in amino acid metabolism and the biosynthesis of antibiotics such as mycomycin and Blasticidin I. Research supported by this grant will test the new mechanistic hypothesis by application of spectroscopic and chemical methods. The aminomutase reactions are also related to vitamin B12; in fact beta-lysine aminomutase is an adenosylcobalamin-dependent enzyme. Lysine 2,3-aminomutase catalyzes a chemically similar 1,2-amino group rearrangement, but does so without a vitamin B12 coenzyme, despite the fat that nearly all rearrangements of this chemical type in living cells involve adenosylcobalamin as an essential coenzyme. The cofactors of lysine 2,3,-aminomutase are iron-sulfur cluster, cobalt(II) and S- adenosylmethionine (AdoMet). Currently available information indicates that AdoMet and the metal cofactors interact chemically to generate an adenosyl- cofactor that has chemical properties in common with adenosycobalamin.a The research supported by this grant will seek to characterize this cofactor by the application of spectroscopic and chemical methods. Thus the central thrust of most of research is the elucidation of new chemical mechanisms in the biological actions of the coenzymatic forms of vitamins B1,B6, and B12. A biochemical problem of recent origin is that of determining the chemical functions of proteins discovered and characterized by genetic methods. A new method for identifying endogenous ligands for such proteins is needed. Such method is being developed for this purposed.

该赠款支持的研究的长期目标是 确定维生素B1的辅酶形成的化学机制 （硫胺素焦磷酸盐，TPP）和维生素B6（吡啶毒素5'-磷酸，PLP） 在反应中作用，无法通过常规机制来解释 以这些辅酶而闻名。 2-乙酰-TPP在代谢中的作用 丙酮酸丙酮酸脱氢酶（PDH）复合物催化的丙酮酸将是 使用化学方法确定。丙酮酸的几种遗传疾病 代谢是由PDH复合物缺陷引起的。标准 PLP辅酶作用的机械范式涉及 辅酶对碳离子中间体的稳定。但是，在一个 这笔赠款支持的主要研究重点，PLP在 赖氨酸2,3-氨基氨基酶催化的赖氨酸的赖氨酸转化为β-赖氨酸PLP 提议通过稳定自由基中间体而不是 Carbanion，。这是与其他PLP的新化学机制 氨基酸酯酶，例如β-赖氨酸突变酶和精氨酸2,3，-aminomutase。这 在 氨基酸代谢和抗生素的生物合成，例如mycomycin 和Blasticidin I.该赠款支持的研究将测试新的 通过应用光谱和化学的机械假设 方法。氨基氨基酶反应也与维生素B12有关。实际上 β-赖氨酸氨基核酶是一种依赖腺苷的酶依赖性酶。赖氨酸 2,3-氨基氨基酶催化化学相似的1,2-氨基 重排，但在没有维生素B12辅酶的情况下这样做，尽管脂肪 在活细胞中，这种化学类型的几乎所有重排 涉及腺苷泡作为必不可少的辅酶。赖氨酸的辅因子 2,3， - 氨基酶是铁硫簇，钴（II）和S- 腺苷硫氨酸（ADOMET）。当前可用的信息表明 Adomet和金属辅因子化学相互作用以产生腺苷 具有化学特性的辅助因子与腺苷的共同特性。 这笔赠款支持的研究将寻求表征该辅助因子 光谱和化学方法的应用。因此中心 大多数研究的推力是阐明新的化学机制 维生素B1，B6和B12的辅酶形式的生物学作用。 最近起源的生化问题是确定化学物质 通过遗传方法发现和特征的蛋白质功能。一个 需要用于鉴定此类蛋白质内源配体的新方法。 目的是为此开发这种方法。